1. Field of the Invention
The present invention relates to a method of detecting the quantity or the presence or absence of a target nucleic acid using a hybridization reaction of a probe nucleic acid immobilized on a substrate and a nucleic acid contained in a sample.
2. Description of the Related Art
As genome sequencing projects advance, for example, detection of specific genes in genome sequences, analysis of single nucleotide polymorphisms (SNPs), and expression analysis of genes have attracted attention as post-genome challenges. Accordingly, in the fields of recent medicine and molecular biology, the importance of analysis methods such as a microarray hybridization method and an in situ hybridization method has been increasing as methods of detecting a target sequence. In the microarray hybridization method and the in situ hybridization method, a nucleic acid probe that specifically binds to a target substance is immobilized on a substrate, and the nucleic acid probe and a sample are hybridized, thus determining the presence of the target substance in the sample.
In general, these hybridization reactions are conducted by dripping a hybridization solution containing a sample on a substrate on which a nucleic acid probe is immobilized. In this process, the substrate is covered with a cover glass so that the hybridization solution does not evaporate. The substrate is then placed in a wet box or a sealed cassette and incubated at a constant temperature for a long time (4 to 50 hours).
However, in the above method of covering with a cover glass, since the hybridization solution on the substrate negligibly moves, the collision frequency between the probe immobilized on the substrate and the sample in the solution is low, resulting in a significantly low hybridization efficiency. Accordingly, it takes a long time to conduct the hybridization reaction, and the method causes a problem in the reliability of data due to uneven hybridization.
In order to increase the hybridization efficiency and to improve the uniformity of hybridization, hybridization ovens having a seesaw-type or roller bottle-type solution-shaking function have been used. However, these hybridization ovens cannot provide a satisfactory effect.
Consequently, hybridization apparatuses aiming at a reduction in the reaction time and an improvement in the uniformity have been recently developed.
An example of such an apparatus is disclosed in U.S. Pat. No. 6,238,910. In this apparatus, a hybridization solution held as a reaction layer is agitated with air (the solution is subjected to a reciprocating shaking), thereby increasing the hybridization efficiency. However, in the mixing by agitation as in this apparatus, it is difficult to maintain the uniformity of the substrate over the entire surface. In addition, once air bubbles are mixed in the apparatus, the air bubbles cannot be eliminated, resulting in an unevenness of hybridization.
Consequently, as disclosed in Japanese Patent Laid-Open No. 2003-315337, a hybridization apparatus in which a reaction solution is circulated in a passage including a substrate has been developed. According to the description of this patent document, the circulation increases the collision frequency between a probe and a sample, and advantages of an increase in the hybridization efficiency and an improvement in the uniformity of hybridization can be achieved.
However, any of these methods is unsatisfactory from the standpoint that the efficiency of the reaction treatment is increased. This is because all the above-described methods are performed on the premise that a complementary strand in the sample solution does not affect the hybridization reaction. That is, these methods require a step of relatively decreasing the complementary strand nucleic acid of the target nucleic acid. Examples of this treatment include an asymmetric polymerase chain reaction (PCR) method using a single-stranded primer and other methods, but these treatments are time-consuming and complex.